The aim of this project is to elucidate the intracellular and extracellular mechanisms that regulate the initiation, and subsequently the direction, of human embryonal carcinoma (EC) cell differentiation, as a model of the mechanisms that regulate cell differentiation in human tumors and in the human embryo. Focusing on the cloned pluripotent human EC cell line NTERA-2 cl.D1 (NT2/D1), we will isolate mutants that do not differentiate in response to the inducers retinoic acid (RA; a natural morphogen) or hexamethylene bisacetamide (HMBA). We will then prepare cell hybrids between the mutants in order to conduct a complementation analysis of the genetic mechanisms by which these inducers are able to initiate differentiation. By retrovirus- mediated transfection, we will introduce various putative regulatory genes (especially homeobox genes, which are developmentally regulated in NT2/D1 cells, and oncogenes) into the parental NT2/D1 cells and into non-responsive mutants, to determine whether and modulate the differentation of these human EC cells and whether any are involved in the mechanisms by which RA and/or HMBA are able to initiate cellular differentiation. In addition, we will continue to analyze and characterize the pathways by which NT2/D1 EC cells differentiate into a variety of derivatives, notably neurons and tentatively identified permitive gut epithelial cells. Immunofluorescence techniques using the FACS and the new ACAS 470 anchored cell analysis and sorting system will be used to follow the progress of differentiation, and to isolate putative intermediate cell types identified operationally on the basis of cell surface antigen expression. Finally, we will use the serum- free medium that we have defined for the growth of human EC cells to analyze the differentiation in the absence of serum and to test the effects of specific growth factors and extracellular matrix molecules (e.g., EGF, FGF, TGF-alpha, TGF-beta, fibronectin, laminin, etc.) on the direction of differentiation of NT2/D1 EC cells after induction with RA or HMBA. We will also use a specific inhibitor of glycosphingolipid synthesis to determine whether any of the developmentally-regulated glycoplipid antigens of NT2/D1 cells play a role in controlling the course of differentiation.